Treatment of joint conditions

ABSTRACT

The invention provides a method of treating a joint condition. The method comprises administering a multi-dose regimen of a pharmaceutical composition comprising a diketopiperazine with amino acid side chains of aspartic acid and alanine (DA-DKP). The invention also provides a method of treating osteoarthritis with multiple doses of a low-molecular weight fraction of human serum albumin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/829,289, filed Aug. 18, 2015, now issued U.S. Pat. No. 9,956,217,which claims the benefit of U.S. Provisional Application No. 62/038,682,filed 18 Aug. 2014, the entirety of each is hereby incorporated byreference.

FIELD OF INVENTION

The invention relates to a method of treating a joint condition. Themethod comprises administering an effective amount of a pharmaceuticalcomposition comprising a diketopiperazine with amino acid side chains ofaspartic acid and alanine (DA-DKP). The invention also provides the useof a pharmaceutical product comprising DA-DKP.

BACKGROUND

Osteoarthritis is the most common form of arthritis, affecting 25 to 35million people in the U.S. Chronic pain and disability of osteoarthritisis initially caused by inflammatory responses in joint cartilage andbone that gradually worsens over time. Symptomatic osteoarthritis of theknee occurs in 10 to 13% of persons aged 60 and over. Kneeosteoarthritis alone increases the risk of loss of mobility, such asneeding assistance walking or climbing stairs, greater than for anyother medical condition in people aged 65 and over.

Current drug treatment for osteoarthritis of the knee is limited toanalgesics, nonsteroidal anti-inflammatory drugs (NSAIDs) andintra-articular steroid injections, all of which have significantlimitations due to adverse effects. Despite these medical treatments,chronic knee osteoarthritis often causes progressive disabilityrequiring total joint replacement. The increasing prevalence ofosteoarthritis of the knee due to aging and obese populations suggests agrowing clinical need for safe and effective local knee treatments thatwill delay and potentially eliminate the need for more extensivesurgical treatments.

SUMMARY OF INVENTION

One embodiment of the present invention is a method of treating a jointcondition by administering to an animal in need treatment an effectiveamount of a pharmaceutical composition that includes DA-DKP in amulti-dose regimen. The joint condition can be a joint disease, such asa degenerative joint disease, for example osteoarthritis. Alternatively,the joint condition can be a joint injury, such as a traumatic injury, apost-operative injury or a repetitive strain injury. Further, the jointcondition can be inflammation.

In the method, the composition can be administered by localadministration, topical administration, or injection, such asintra-articular injection. If administered by intra-articular injection,the composition can have a concentration of DA-DKP from about 50 μM toabout 350 μM.

In the method, the composition can further comprise N-acetyl-tryptophan(NAT), caprylic acid, caprylate or combinations thereof. In thisembodiment, the composition can have a concentration of NAT, caprylicacid, caprylate or combinations thereof from about 4 mM to about 20 mM.In a further embodiment, the DA-DKP can be in a composition prepared byremoving albumin from a solution of a human serum albumin composition,such as by treating a human serum albumin composition by a separationmethod selected from ultrafiltration, sucrose gradient centrifugation,chromatography, salt precipitation, and sonication. In a particularembodiment, the step of removing can include passing a human serumalbumin composition over an ultrafiltration membrane with a molecularweight cut off that retains the albumin, and wherein the resultingfiltrate comprises DA-DKP, such as by use of an ultrafiltration membranethat has a molecular weight cutoff of less than 50 kDa, less than 40kDa, less than 30 kDa, less than 20 kDa, less than 10 kDa, less than 5kDa or less than 3 kDa.

In a further embodiment, the pharmaceutical composition can furtherinclude a second drug selected from an analgesic, an anti-inflammatorydrug, and combinations thereof.

In the method, the number of doses in the multi-dose regimen can bebetween 2 and 10, between 2 and 8, between 2 and 6, between 2 and 4 orbe 3. Further, the time between doses can be between 2 days and 6 weeksbetween 2 days and 5 weeks, between 2 days and 4 weeks, between 2 daysand 3 weeks, between 1 week and 3 weeks, or 2 weeks.

In a preferred embodiment, the present invention is a method of treatingosteoarthritis by administering, via intra-articular injection into anaffected joint, a first dose, a second dose, and a third dose. In thisembodiment, each of the first dose, the second dose, and the third dosecomprises 4 mL of a <5000 MW fraction of human serum albumin, andfurther, the second dose is administered two weeks after the first doseand the third dose is administered two weeks after the second dose.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the mean percent change in WOMAC A pain score for thetreatment described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of treating a joint condition byadministering a composition comprising DA-DKP in a multi-dose regimen.The treatment comprises administering an effective amount of apharmaceutical composition comprising aspartyl-alanyl diketopiperazine(DA-DKP) to an individual having a need thereof. DA-DKP has multipleanti-inflammatory and immune modulating effects including inhibition ofmultiple pro-inflammatory cytokines, chemokines and signaling moleculesat the transcription level, inhibition of the migration and adhesion ofT-cells and monocytes, activity at the G-coupled protein receptor level,activity on actin-dependent cytoskeletal events, reduction in vascularpermeability and inhibition of inflammation induced by plateletactivating factor. As described in more detail below, the effects ofDA-DKP on joint conditions have been found to be unexpectedly longlasting and in some studies were found to increase in time as comparedto the use of steroids.

The invention also provides for a pharmaceutical product comprising aDA-DKP composition. The DA-DKP of the product can be prepared byremoving albumin from a solution of human serum albumin.

As used herein, the term “joint condition” refers to any disease,illness, or injury of a joint. Examples of joint conditions include, butare not limited to, acute diseases, chronic diseases, refractorydiseases, progressive diseases (including degenerative diseases),traumatic injuries, repetitive strain injuries, toxic injuries,post-operative conditions, and inflammation with or without structuraldamage.

A degenerative joint disease is a gradual deterioration of the articularcartilage that covers joints. A degenerative joint disease(osteoarthritis) is a noninfectious progressive disorder of theweightbearing joints. The normal articular joint cartilage is smooth,white, and translucent. It is composed of cartilage cells (chondrocytes)imbedded in a sponge-like matrix made of collagen, proteinpolysaccharides, and water. With early primary arthritis, the cartilagebecomes yellow and opaque with localized areas of softening androughening of the surfaces. As degeneration progresses, the soft areasbecome cracked and worn, exposing bone under the cartilage. The bonethen begins to remodel and increase in density while any remainingcartilage begins to fray. Eventually, osteophytes (spurs of new bone)covered by cartilage form at the edge of the joint. As mechanical wearincreases, the cartilage needs repairing. The cartilage cells are unableto produce enough of the sponge-like matrix and therefore the damagedcartilage cannot repair itself. The cartilage has no blood supply toenhance healing. The majority of degenerative joint disease is theresult of mechanical instabilities or aging changes within the joint.This includes old age degenerative arthritis and, in youngerindividuals, may be the result of injuries, bruises, abnormal jointconfiguration (i.e. hip dysplasia), or mechanical wear from anteriorcruciate ligament rupture, patellar luxation, or osteochondritisdissecans, for example. Degenerative joint disease can occur at anyjoint in the body, including without limitation, knee, hip, shoulder,hand and spine.

Conventional pharmaceutical therapies for joint conditions includeacetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDS), narcotics,and corticosteroids.

“Treat” is used herein to mean to reduce (wholly or partially) thesymptoms, duration or severity of a condition.

The pharmaceutical composition comprising DA-DKP of the invention isadministered to an animal in need of treatment. Preferably, the animalis a mammal, such as a rabbit, goat, dog, cat, horse or human. Effectivedosage amounts can vary with the severity of the disease or condition,the route(s) of administration, the duration of the treatment, theidentity of any other drugs being administered to the animal, the age,size and species of the animal, and like factors known in the medicaland veterinary arts.

The composition of the present invention comprising DA-DKP may beadministered to an animal patient for therapy by any suitable route ofadministration, including locally, parenterally (e.g., injection,intra-articular injection, intravenously, intraspinally,intraperitoneally, subcutaneously, or intramuscularly), transdermally,and topically. A preferred route of administration is intra-articularinjection.

The composition of the present invention can be a pharmaceuticalsolution having a DA-DKP concentration range with a lower endpoint ofabout 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110μM, about 120 μM, about 130 μM, about 140 μM, about 150 μM, about 160μM, about 170 μM, about 180 μM, about 190 μM, about 200 μM, about 210μM, about 220 μM, about 230 μM, about 240 μM, about 240, about 250 μM,about 260 μM, about 270 μM, about 280 μM, about 290 μM, about 300 μM,about 310, about 320 μM, about 330 μM, about 340 μM, about 350 μM, about360 μM, about 370 μM, about 380 μM, about 390 or about 400 μM.

The composition of the present invention can be a pharmaceuticalsolution having a DA-DKP concentration range with an upper endpoint ofabout 600 μM, about 580 μM, about 570 μM, about 560 μM, about 550 μM,about 540 μM, about 530 μM, about 520 μM, about 510 μM, about 500 μM,about 490 μM, about 480 μM, about 470 μM, about 460 μM, about 450 μM,about 440 μM, about 430 μM, about 420 μM, about 410 μM, about 400 μM,about 390 μM, about 380 μM, about 370 μM, about 360 μM, about 350, about340 μM, about 330 μM, about 320 μM, about 310 μM, about 300 μM, about290 μM, about 280, about 270 μM, about 260 μM, about 250 μM, about 240μM, about 230 μM, about 220 μM, about 210 μM, or about 200 μM.

An effective amount of DA-DKP in the composition of the presentinvention for treating a joint condition can be a range with a lowerendpoint of about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg,about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about85 μg, about 90 μg, about 95 μg, about 100 μg, about 110 μg, about 120μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 170μg, about 180 μg, about 190 μg, about 200 μg, about 210 μg, about 220μg, about 230 μg, about 240 μg, about 250 μg, about 260 μg, about 270μg, about 280 μg, about 290 μg, about 300 μg, about 310 μg, about 320μg, about 330 μg, about 340 μg, about 350 μg, about 360 μg, about 370μg, about 380 μg, about 390 μg, about 400 μg, about 425 μg, about 450μg, about 475 μg or about 500 μg. In addition, an effective amount ofDA-DKP in the composition of the present invention for treating a jointcondition can be a range with upper endpoint of about 500 μg, about 490μg, about 480 μg, about 470 μg, about 460 μg, about 450 μg, about 440μg, about 430 μg, about 420 μg, about 410 μg, about 400 μg, about 390μg, about 380 μg, about 370 μg, about 360 μg, about 350 μg, about 340μg, about 330 μg, about 320 μg, about 310 μg, about 300 μg, about 290μg, about 280 μg, about 270 μg, about 260 μg, about 250 μg, about 240μg, about 230 μg, about 220 μg, about 210 μg, about 200 μg, about 190μg, about 180 μg, about 170 μg, about 160 μg, about 150 μg, about 140μg, about 130 μg, about 120 μg, about 110 μg, about 100 μg, about 90 μg,about 80 μg, about 70 μg, about 60 μg, about 50 μg, about 40 μg, about30 μg, or about 20 μg.

In embodiments where DA-DKP is administered and a low molecular weightfraction of human serum albumin, such as a <5000 MW fraction asdescribed below and as is exemplified by Ampion™, the dose amountadministered to a patient can be between about 1 mL and about 20 mL,between about 1 mL and about 15 mL, between about 1 mL and about 10 mL,between about 1 mL and about 8 mL, between about 2 mL and about 6 mL,between about 3 mL and about 5 mL or about 4 mL.

Dosage forms for the topical or transdermal administration of compoundsof the invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, and drops. The active ingredient maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any buffers, or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to theactive ingredient, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof powders and sprays can contain, in additionto the active ingredient, excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder ormixtures of these substances. Sprays can additionally contain customarypropellants such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of compounds of the invention to the body. Such dosage formscan be made by dissolving, dispersing or otherwise incorporating one ormore compounds of the invention in a proper medium, such as anelastomeric matrix material. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate of such fluxcan be controlled by either providing a rate-controlling membrane ordispersing the compound in a polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministrations comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drug isaccomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

Kits comprising the pharmaceutical products of the present invention arealso provided. The kits can comprise a DA-DKP composition formulated foradministration by injection. The DA-DKP can be prepared as describedherein, such as by removing albumin from a solution of a human albumincomposition. The kits may contain unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use. Thekits may also be stored in a condition, wherein the contents are readyfor direct use or injection.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The pharmaceuticalcompositions of the invention comprise a compound or compounds of theinvention as an active ingredient in admixture with one or morepharmaceutically-acceptable carriers and, optionally, with one or moreother compounds, drugs or other materials. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the animal.Pharmaceutically-acceptable carriers are well known in the art.Regardless of the route of administration selected, the compounds of thepresent invention are formulated into pharmaceutically-acceptable dosageforms by conventional methods known to those of skill in the art. See,e.g., Remington's Pharmaceutical Sciences.

The composition of the present invention can further compriseN-acetyl-tryptophan (NAT), caprylic acid, caprylate or combinationsthereof. Preferably, the composition can comprise NAT. Compositions ofthe present invention having NAT, caprylic acid, caprylate orcombinations thereof can be a pharmaceutical composition having NAT,caprylic acid, caprylate or combinations thereof concentration rangewith a lower endpoint of about 1 mM, about 2 mM, about 3 mM, about 4 mM,about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM,about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM. Inaddition, compositions of the present invention having NAT, caprylicacid, caprylate or combinations thereof can be a pharmaceuticalcomposition having a NAT, caprylic acid, caprylate or combinationsthereof concentration range with an upper endpoint of about 40 mM, about39 mM, about 38 mM, about 37 mM, about 36 mM, about 35 mM, about 34 mM,about 33 mM, about 32 mM, about 31 mM, about 30 mM, about 29 mM, about28 mM, about 27 mM, about 26 mM, about 25 mM, about 24 mM, about 23 mM,about 22, or about 21 mM. Preferably, the concentration range is about 4mM to about 20 mM.

In addition, the composition of the present invention may also comprisea second drug such as an analgesic (such as lidocaine or paracetoamol),an anti-inflammatory (such as bethamethasone, non-steroidanti-inflammatory drugs (NSAIDs), acetaminophen, ibuprofen, naproxen),and/or other suitable drugs.

Methods of making diketopiperazines, such as DA-DKP, are well known inthe art, and these methods may be employed to synthesize thediketopiperazines of the invention. See, e.g., U.S. Pat. Nos. 4,694,081,5,817,751, 5,990,112, 5,932,579 and 6,555,543, U.S. Patent ApplicationPublication Number 2004/0024180, PCT applications WO 96/00391 and WO97/48685, and Smith et al., Bioorg. Med. Chem. Letters, 8, 2369-2374(1998), the complete disclosures of which are incorporated herein byreference.

For instance, diketopiperazines, such as DA-DKP, can be prepared byfirst synthesizing dipeptides. The dipeptides can be synthesized bymethods well known in the art using L-amino acids, D-amino acids or acombination of D- and L-amino acids. Preferred are solid-phase peptidesynthetic methods. Of course, dipeptides are also available commerciallyfrom numerous sources, including DMI Synthesis Ltd., Cardiff, UK (customsynthesis), Sigma-Aldrich, St. Louis, Mo. (primarily custom synthesis),Phoenix Pharmaceuticals, Inc., Belmont, Calif. (custom synthesis),Fisher Scientific (custom synthesis) and Advanced ChemTech, Louisville,Ky.

Once the dipeptide is synthesized or purchased, it is cyclized to form adiketopiperazine. This can be accomplished by a variety of techniques.For example, U.S. Patent Application Publication Number 2004/0024180describes a method of cyclizing dipeptides. Briefly, the dipeptide isheated in an organic solvent while removing water by distillation.Preferably, the organic solvent is a low-boiling azeotrope with water,such as acetonitrile, allyl alcohol, benzene, benzyl alcohol, n-butanol,2-butanol, t-butanol, acetic acid butylester, carbon tetrachloride,chlorobenzene chloroform, cyclohexane, 1,2-dichlorethane, diethylacetal,dimethylacetal, acetic acid ethylester, heptane, methylisobutylketone,3-pentanol, toluene and xylene. The temperature depends on the reactionspeed at which the cyclization takes place and on the type ofazeotroping agent used. The reaction is preferably carried out at50-200° C., more preferably 80-150° C. The pH range in which cyclizationtakes place can be easily determined by the person skilled in the art.It will advantageously be 2-9, preferably 3-7.

When one or both of the amino acids of the dipeptide has, or isderivatized to have, a carboxyl group on its side chain (e.g., asparticacid or glutamic acid), the dipeptide is preferably cyclized asdescribed in U.S. Pat. No. 6,555,543. Briefly, the dipeptide, with theside-chain carboxyl still protected, is heated under neutral conditions.Typically, the dipeptide will be heated at from about 80° C. to about180° C., preferably at about 120° C. The solvent will be a neutralsolvent. For instance, the solvent may comprise an alcohol (such asbutanol, methanol, ethanol, and higher alcohols, but not phenol) and anazeotropic co-solvent (such as toluene, benzene, or xylene). Preferably,the alcohol is butan-2-ol, and the azeotropic co-solvent is toluene. Theheating is continued until the reaction is complete, and such times canbe determined empirically. Typically, the dipeptide will be cyclized byrefluxing it for about 8-24 hours, preferably about 18 hours. Finally,the protecting group is removed from the diketopiperazine. In doing so,the use of strong acids (mineral acids, such as sulfuric or hydrochloricacids), strong bases (alkaline bases, such as potassium hydroxide orsodium hydroxide), and strong reducing agents (e.g., lithium aluminumhydride) should be avoided, in order to maintain the chirality of thefinal compound.

Dipeptides made on solid phase resins can be cyclized and released fromthe resin in one step. See, e.g., U.S. Pat. No. 5,817,751. For instance,the resin having an N-alkylated dipeptide attached is suspended intoluene or toluene/ethanol in the presence of acetic acid (e.g., 1%) ortriethylamine (e.g., 4%). Typically, basic cyclization conditions arepreferred for their faster cyclization times.

Other methods of cyclizing dipeptides and of making diketopiperazinesare known in the art and can be used in the preparation ofdiketopiperazines useful in the practice of the invention. See, e.g.,those references listed above. In addition, many diketopiperazinessuitable for use in the present invention can be made as described belowfrom proteins and peptides. Further, diketopiperazines for use in thepractice of the invention can be obtained commercially from, e.g., DMISynthesis Ltd., Cardiff, UK (custom synthesis).

The DA-DKP composition and/or products of the present invention can beprepared from solutions containing DA-DKP, including from thecommercially-available pharmaceutical compositions comprising albumin,such as human serum albumin, by well known methods, such asultrafiltration, size-exclusion chromatography, affinity chromatography(e.g., using a column of beads having attached thereto an antibody orantibodies directed to the desired diketopiperazine(s) or an antibody orantibodies directed to the truncated protein or peptide), anion exchangeor cation exchange), sucrose gradient centrifugation, chromatography,salt precipitation, or sonication, that will remove some or all of thealbumin in the solution. The resultant DA-DKP-containing compositionand/or product can be used and incorporated into pharmaceuticalcompositions as described above.

Using an ultrafiltration separation method, a human serum albumincomposition can be passed over an ultrafiltration membrane having amolecular weight cut-off that retains the albumin while the DA-DKPpasses into the resulting filtrate or fraction. This filtrate maycomprise components having molecular weights less than about 50 kDA,less than about 40 kDa, less than 30 kDa, less than about 20 kDa, lessthan about 10 kDa, less than about 5 kDa, or less than about 3 kDa.Preferably, the filtrate comprises components having molecular weightsless than about 5 Da (also referred to as “<5000 MW”). This <5000 MWfraction or filtrate contains DA-DKP which is formed after the dipeptideaspartate-alanine is cleaved from albumin and subsequently cyclized intothe diketopiperazine.

Physiologically-acceptable salts of the DA-DKP of the invention may alsobe used in the practice of the invention. Physiologically-acceptablesalts include conventional non-toxic salts, such as salts derived frominorganic acids (such as hydrochloric, hydrobromic, sulfuric,phosphoric, nitric, and the like), organic acids (such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,glutamic, aspartic, benzoic, salicylic, oxalic, ascorbic acid, and thelike) or bases (such as the hydroxide, carbonate or bicarbonate of apharmaceutically-acceptable metal cation or organic cations derived fromN,N-dibenzylethylenediamine, D-glucosamine, or ethylenediamine). Thesalts are prepared in a conventional manner, e.g., by neutralizing thefree base form of the compound with an acid.

The present invention involves a method of treating a joint conditionthat includes administering to an animal in need thereof an effectiveamount of a pharmaceutical composition comprising DA-DKP in a multi-doseregimen. It has been surprisingly found that significant improvements inpain reduction and improved function can be achieved by the use of amulti-dose regimen to administer pharmaceutical compositions of thepresent invention as compared to a single dose regimen. U.S. Pat. No.8,980,834 to Bar-Or et al. (“Bar-Or”), assigned to AmpioPharmaceuticals, Inc., discloses the treatment of osteoarthritis byinjection of a <5000 MW fraction of human serum albumin (referred to asAmpion™). Bar-Or does not disclose a multi-dose regimen and in fact,suggests that because of the long lasting effect of the treatment,single dose administration is sufficient for time periods up to sixmonths. Without intending to be bound by theory, the improved effect ofa multi-dose regimen is believed to be achieved by more than a simpleincrease in the amount of active composition being administered. Asshown below in Example 1, no benefit was seen by increasing a singledose administration from 4 mL of Ampion™ to 10 mL. However, when three 4mL doses of Ampion™ (total of 12 mL) are administered two weeks apart, asignificant benefit was seen as shown in Example 2. Thus, there isbelieved to be at least some effect achieved by prolonging exposure ofthe joint to the treatment by the extended multi-dose regimen that isindependent of the total amount of active composition beingadministered.

A multi-dose regimen refers to administration of pharmaceuticalcompositions of the present invention to a patient in multiple dosesthat are spread apart in time. Two important variables within amulti-dose regimen are the number of doses and the timing between doses.In the present invention, the number of doses is more than two and canbe from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, or 3. In thepresent invention, the timing between any two doses can be from 2 daysto 6 weeks, from 2 days to 5 weeks, from 2 days to 4 weeks, from 2 daysto 3 weeks, from 1 week to 2 weeks, or can be about 2 weeks.

The present invention can provide significant improvements in pain fromjoint conditions such as osteoarthritis and particularly in the kneejoint, as compared to single administration of the treatment. Pain canbe evaluated on a number scales and one suitable pain scale is WOMAC A.The present invention can improve WOMAC A scores or other pain scales by10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% over baseline scores.

This embodiment of the present invention can provide significantimprovements in joint function and particularly function of the kneejoint. Function of joints can be evaluated on a number scales and onesuitable scale is WOMAC C. The present invention can improve WOMAC Cscores or other joint function scales by 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% over baseline scores.

As used herein, “a” or “an” means one or more.

As used herein, “comprises” and “comprising” include within their scopeall narrower terms, such as “consisting essentially of” and “consistingof” as alternative embodiments of the present invention characterizedherein by “comprises” or “comprising”. In regard to use of “consistingessentially of”, this phrase limits the scope of a claim to thespecified steps and materials and those that do not materially affectthe basic and novel characteristics of the invention disclosed herein.

Additional objects, advantages and novel features of the presentinvention will become apparent to those skilled in the art byconsideration of the following non-limiting examples. The followingexperimental results are provided for purposes of illustration and arenot intended to limit the scope of the invention.

EXAMPLES Example 1

A randomized, placebo-controlled, double-blind study was performed toevaluate the efficacy and safety of two doses of intra-articular (IA)injection of Ampion™ in adults with pain due to osteoarthritis of theknee (OAK).

Primary Objective

To evaluate whether the efficacy of 10 mL Ampion™ versus 10 mL placebois greater than the efficacy of 4 mL Ampion™ versus 4 mL placebo IAinjection in improving knee pain, when administered to patientssuffering from OAK.

Study Subjects

Study subjects were male and female adult patients who were 40 years to85 years old (inclusive). Eligible patients were ambulatory butsuffering from moderate to moderately severe pain from OAK in the indexknee, as evidenced by a rating of at least 1.5 on the WOMAC Index 3.1,five-point Likert Pain Subscale at screening. Patients must have had anindex knee that was symptomatic for greater than six months with aclinical diagnosis of OAK, which was supported by radiological evidence(Kellgren-Lawrence (KG) Grade 2 to 4) obtained no more than six monthsprior to screening. Patients were also required to have moderate tomoderately severe OAK pain in the index knee even with chronic dosing ofNSAID in the four weeks prior to screening. Patients could not havetaken analgesia (including acetaminophen) for twelve hours prior to anefficacy measure.

Treatments

The test product was Ampion™, 4 mL or 10 mL, administered as a single IAinjection in the knee. The control product was saline placebo, 4 mL or10 mL, administered as a single IA injection in the knee. Patients whomet the study entry criteria were randomly assigned in a 1:1:1:1 ratioto the following four study arms: 4 mL Ampion™, 4 mL placebo, 10 mLAmpion™, and 10 mL placebo.

The clinical effects of treatment on OAK pain were evaluated using theWOMAC and the Patient's Global Assessment (PGA) at office visits at sixand twelve weeks, and optionally at twenty weeks, and during telephonecontacts at two, four, eight, and ten weeks. The total duration of thestudy was twelve weeks, optionally up to twenty weeks, excluding thescreening period of up to four weeks before Day 0.

Primary Efficacy Endpoint

The primary efficacy endpoint of this study was the change in the WOMACA pain subscore on the five-point Likert scale between baseline (Day 0)and Week 12.

Secondary Efficacy Endpoints

The secondary efficacy endpoints of this study were:

-   -   Change in WOMAC A pain subscore between baseline and Weeks 2, 4,        6, 8, and 10, and optionally Week 20;    -   Change in WOMAC B stiffness subscore between baseline and Weeks        2, 4, 6, 8, 10, and 12;    -   Change in WOMAC C physical function subscore between baseline        and Weeks 2, 4, 6, 8, 10, and 12, and optionally Week 20;    -   Change in PGA between baseline and Weeks 6, 8, 10, and 12, and        optionally Week 20;    -   Response status based on the OMERACT-OARSI criteria at Weeks 2,        4, 6, 8, 10, and 12;    -   Change in WOMAC A pain subscore average of questions 1 and 2        (pain with movement) between baseline and Weeks 2, 4, 6, 8, 10,        and 12;    -   Change in WOMAC A pain subscore average of questions 3 through 5        (pain during rest) between baseline and Weeks 2, 4, 6, 8, 10,        and 12;    -   Use of rescue analgesia (amount of acetaminophen used); and    -   Incidence and severity of TEAEs.        Safety Assessments

Safety assessments included collection of concomitant medication data,physical examinations, vital sign measurements, clinical laboratorymeasurements, and monitoring of TEAEs.

Demographic and Baseline Characteristics

The study population was representative of the population that would beexpected to receive treatment with Ampion™. Baseline characteristicswere similar across treatment groups: the majority of patients in eachgroup were White and female, and had a median age of approximately 62years.

Efficacy Results

Subjects receiving Ampion™ achieved significantly greater reduction inWOMAC A pain subscores (improvement) from baseline to Week 12 comparedwith those receiving placebo (P=0.0038), representing approximately 42%reduction in pain from baseline. Moreover, this improvement in pain wasmore pronounced in patients with more severe OAK disease; for example,patients with KG Grades 3 and 4 disease had greater improvement in paincompared with placebo at Week 12. Patients receiving Ampion™ alsoachieved significantly greater improvement in function (WOMAC C) frombaseline to Week 12 compared with patients receiving placebo (P=0.04).Additionally, patients receiving Ampion™ also experienced significantlygreater improvement in overall quality of life measures, as measured bythe PGA, from baseline to Week 12 compared with patients receivingplacebo (P=0.01). These clinically and statistically significantimprovements in pain, function, and overall quality of life measureswere observed after only a single Ampion™ IA injection into the knee.

TABLE 1 WOMAC A pain subscore - mean and change from baseline to Week 12Randomized Arms Combined Arms Placebo Placebo Ampion Ampion PlaceboAmpion 4 mL 10 mL 4 mL 10 mL 4 + 10 mL 4 + 10 mL (N = 83) (N = 81) (N =83) (N = 82) (N = 164) (N = 165) P-value* BASELINE n 83 81 83 82 164 165Mean (SD) 2.32 (0.548) 2.23 (0.602) 2.22 (0.490) 2.19 (0.512) 2.27(0.575) 2.20 (0.500) Median 2.20 2.00 2.20 2.20 2.20 2.20 IQR 1.80, 2.601.80, 2.60 1.80, 2.60 1.80, 2.60 1.80, 2.60 1.80, 2.60 Min, Max 1.4, 4.01.0, 3.6 1.2, 3.4 1.2, 3.4 1.0, 4.0 1.2, 3.4 Not Reported 0 0 0 0 0 0WEEK 12 n 83 81 83 82 164 165 Mean (SD) 1.61 (0.759) 1.50 (0.875) 1.28(0.790) 1.27 (0.764) 1.55 (0.818) 1.28 (0.775) Median 1.60 1.60 1.201.20 1.60 1.20 IQR 1.00, 2.00 1.00, 2.20 0.60, 2.00 0.80, 1.80 1.00,2.20 0.60, 2.00 Min, Max 0.2, 3.6 0.0, 3.8 0.0, 2.8 0.0, 3.2 0.0, 3.80.0, 3.2 Not Reported 0 0 0 0 0 0 Change (Week 12) n Change 83 81 83 82164 165 0.0038 Mean Change −0.71 (0.752) −0.73 (0.964) −0.93 (0.764)−0.92 (0.791) −0.72 (0.860) −0.93 (0.775) (SD) 95% CI of −0.871, −0.543−0.946, −0.520 −1.099, −0.766 −1.093, −0.746 −0.852, −0.587 −1.045,−0.807 Mean Change

In the overall group of subjects that attended the Week 20 visit, therewas no statistical significance in mean change from baseline in WOMAC Apain subscores; however there was a statistically significantimprovement in the KL Grade 4 subset (P=0.036).

TABLE 2 WOMAC A pain subscore in Kellgren-Lawrence (KL) Grade 4subjects - mean and change from baseline to week 20 Placebo PlaceboAmpion Ampion Placebo Ampion KL 4 Subset 4 mL 10 mL 4 mL 10 mL 4 mL + 10mL 4 mL + 10 mL P-value BASELINE (Randomization) n 45 44 52 52 89 104Mean (SD) 2.28 (0.552) 2.22 (0.546) 2.16 (0.474 ) 2.22 (0.501) 2.25(0.547) 2.19 (0.486) Median 2.20 2.20 2.00 2.20 2.20 2.20 IQR 1.80, 2.601.80, 2.50 1.80, 2.60 2.00, 2.60 1.80, 2.60 1.80, 2.60 Min, Max 1.6, 4.01.6, 3.6 1.2, 3.4 1.2, 3.4 1.6, 4.0 1.2, 3.4 Not Reported 0 0 0 0 0 0WEEK 20 n 14 13 11 10 27 21 Mean (SD) 1.89 (0.655) 1.92 (0.630) 1.22(0.855) 1.70 (0.492) 1.90 (0.631) 1.45 (0.732) Median 1.90 2.00 1.001.70 2.00 1.40 IQR 1.20, 2.40 1.80, 2.40 0.60, 2.00 1.20, 2.20 1.20,2.40 1.00, 2.00 Min, Max 0.8, 2.8 0.6, 3.0 0.2, 2.6 1.0, 2.4 0.6, 3.00.2, 2.6 Not Reported 0 0 0 0 0 0 Change (Week 20) n Change 14 13 11 1027 21 Mean Change −0.69 (0.713) −0.32 (0.893) −1.09 (1.104) −0.32(0.598) −0.51 (0.810) −0.72 (0.962) 0.0360 (SD) 95% CI of −1.098, −0.274−0.863, 0.216  −1.833, −0.349 −0.748, 0.108  −0.832, −0.191 −1.162,−0.286 Mean ChangeConclusions

The results of this study establish the safety and efficacy of Ampion™for reduction of pain at twelve weeks after a single IA injection in theknee of patients with OAK. Both 4 mL and 10 mL doses of Ampion™ aresafe, efficacious, and well tolerated. In the absence of a difference inefficacy for the 4 mL and 10 mL Ampion™ doses, the lower dose of 4 mLwas to be evaluated in further studies. The reduction in the mean WOMACA pain subscore of KL Grade 4 patients in the combined 4 mL and 10 mLarms at 20 weeks was about 34%. In the overall group of patientsevaluated at week 20, including patients in addition to KL Grade 4,there was no statistically significant change.

Example 2

A prospective Phase I/II study was performed to evaluate the safety andefficacy of three intra-articular injections of Ampion™ (4 mL)administered two weeks apart in adults with pain due to osteoarthritisof the knee.

Primary Objectives

The primary objective of Phase I was to evaluate the safety of Ampion™ 4mL administered as three IA injections, two weeks apart, in patientssuffering from OAK of the knee from baseline to Week 20.

The primary objective of Phase II was to evaluate the safety andefficacy of Ampion™ 4 mL versus placebo injection from baseline to Week20, when administered as 3 IA injections, in improving knee pain inpatients suffering from OAK of the knee.

Study Subjects

For both the Phase I and Phase II studies, subjects were male and femaleadult patients who were 40 years to 85 years old (inclusive) with OAKknee pain. Eligible patients were required to be ambulatory and theindex knee must have been symptomatic for greater than six months with aclinical diagnosis of OAK supported by radiological evidence (KL Grades2 to 4) acquired at screening. Patients must have had moderate tomoderately severe OAK pain in the index knee (rating of at least 1.5 onthe WOMAC Index 3.1 five-point Likert Pain Subscale) assessed atscreening and confirmed at randomization. The moderate to moderatelysevere OAK pain in the index knee must have been present even if chronicdoses of NSAIDs, which had not changed in the four weeks prior toscreening, had been or were being used. Patients could not have takenanalgesia for twelve hours prior to an efficacy measure. It wasrecommended that patients should have a WOMAC five-point Likert PainSubscale score of less than 1.5 in the contralateral knee, which wasassessed at screening.

In Phase I, a total of seven patients were to be enrolled, andenrollment of KL Grade 2 patients was limited to no more than twopatients. The medical monitor conducted a safety evaluation after allseven patients had completed the Week 4 follow-up evaluation. Enrollmentwas to be initiated in Phase II if no serious drug-related AEs orunanticipated drug-related AEs were observed. Pending safety review, theseven patients were to continue in the Phase I study until Week 52.

In Phase II, approximately forty patients were to be enrolled andrandomized 1:1 across the two study arms (twenty patients per studyarm). Enrollment of KL Grade 2 patients was limited to 25% of totalenrollment in Phase II, i.e. eight patients total, randomized 1:1 acrossthe two study arms.

Treatments

The test product for both Phase I and Phase II was Ampion™, and thecontrol product for the Phase II study was saline placebo.

For Phase I, patients who met the study entry criteria received three IAinjections of 4 mL Ampion™ in the knee at baseline (Day 0) and at Weeks2 and 4. For Phase II, patients who met the study entry criteria wererandomly assigned in a 1:1 ratio to the 4 mL Ampion™ study arm or the 4mL placebo study arm. Subjects received three IA injections in the kneeof study medication (Ampion™ or placebo) at baseline (Day 0) and atWeeks 2 and 4.

Subjects attended in-clinic visits at Week 6, Week 12, Week 20, Week 24,and Week 52. The maximum study duration for each patient was 52 weeks,excluding a screening period of up to four weeks.

Endpoints

In Phase I, the incidence and severity of TEAEs and SAEs was determined.

The primary efficacy endpoint of Phase II was the change in the WOMAC Apain subscore by the five-point Likert scale from baseline (Day 0) toWeek 20. Secondary endpoints of Phase II were:

-   -   Change in WOMAC A pain subscore from baseline (Day 0) to Weeks        2, 4, 6, 12, 24, and 52;    -   Change in WOMAC C physical function subscore from baseline        (Day 0) to Weeks 2, 4, 6, 12, 20, 24, and 52;    -   Change in PGA from baseline (Day 0) to Weeks 2, 4, 6, 12, 20,        24, and 52;    -   Use of rescue analgesia (amount of acetaminophen used) through        Week 20; and    -   Incidence and severity of TEAEs        Safety Assessments

Safety assessments included recording TEAEs at all in-clinic visits andthe 24-hour post-injection telephone contact calls; physical examinationand vital sign recordings at in-clinic visits at baseline (Day 0) and atWeeks 2, 4, 6, 12, 20, 24, and 52); and standard laboratory tests.

Exposure

In Phase I, seven subjects were enrolled and received all threeinjections of Ampion™. In Phase II, forty subjects (twenty Ampion™,twenty placebo) were enrolled and received all three injections of studydrug.

Demographic and Baseline Characteristics

Baseline characteristics were similar across treatment groups: themajority of patients in each treatment group were White and female, witha median age of 62.5 years, and had KL Grade 3 (58%) or Grade 4 (38%).

Efficacy Results

Subjects receiving Ampion™ achieved significantly greater reduction inWOMAC A pain subscores (improvement) from baseline to Week 20 comparedwith those receiving placebo (P=0.0231). This represents a reduction inmean pain of approximately 64% from baseline in the Ampion™ arm comparedwith a reduction of approximately 40% in the placebo arm (P=0.0313), asillustrated in FIG. 1.

TABLE 3 WOMAC A pain subscore - mean and change form baseline to Week 20Ampion Placebo 4 mL 4 mL (N = 20) (N = 20) P-value BASELINE N 19 19 Mean(SD) 2.26 (0.46) 2.18 (0.45) Median 2.40 2.20 Min, Max 1.6, 3.0 1.5, 3.0WEEK 20 N 19 19 0.5960 Mean (SD) 0.85 (0.88) 1.34 (0.73) Median 0.401.40 Min, Max 0.0, 3.0 0.0, 2.2 Change (Week 20) N Change 19 19 0.0231Mean Change (SD) −1.41 (0.81) −0.85 (0.64) 95% CI difference (0.10,1.03) for Mean Change

Patients receiving Ampion™ demonstrated no significant differences inWOMAC A pain sub score from baseline at Weeks 2, 4, 6, 12, or 24compared with patients receiving placebo.

Conclusions

The results of this study establish the safety and efficacy of Ampion™for reduction of pain at twenty weeks after three IA injections, twoweeks apart, in the knee of patients with OAK. The 4 mL dose of Ampion™was safe, efficacious, and well tolerated.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing exemplary claims.

What is claimed is:
 1. A method of treating a joint condition comprisingadministering to an animal with a Kellgren-Lawrence Grade 4 pain scorean effective amount of a pharmaceutical composition comprising DA-DKPprepared by removing albumin from a solution of a human serum albumincomposition in a multi-dose regimen, wherein the number of doses in themulti-dose regimen is between 2 and 6 doses per 52 weeks.
 2. The methodof claim 1, wherein the joint condition is a joint disease.
 3. Themethod of claim 2, wherein the joint disease is a degenerative jointdisease.
 4. The method of claim 3, wherein the degenerative jointdisease is osteoarthritis.
 5. The method of claim 1, wherein the jointcondition is a joint injury.
 6. The method of claim 5, wherein the jointinjury is at least one of a traumatic injury and a post-operativeinjury.
 7. The method of claim 5, wherein the joint injury is arepetitive strain injury.
 8. The method of claim 1, wherein the jointcondition is inflammation.
 9. The method of claim 1, wherein thecomposition is administered by an administration route selected from thegroup consisting of local administration, topical administration, andinjection.
 10. The method of claim 9, wherein administration byinjection is by intra-articular injection.
 11. The method of claim 10,wherein the composition administered by intra-articular injection is acomposition having a concentration of DA-DKP from about 50 μM to about350 μM.
 12. The method of claim 1, wherein the composition furthercomprises N-acetyl-tryptophan (NAT), caprylic acid, caprylate orcombinations thereof.
 13. The method of claim 10, wherein thecomposition is a composition having a concentration of NAT, caprylicacid, caprylate or combinations thereof from about 4 mM to about 20 mM.14. The method of claim 1, wherein the step of removing the albumincomprises treating the human serum albumin composition by a separationmethod selected from the group consisting of ultrafiltration, sucrosegradient centrifugation, chromatography, salt precipitation, andsonication.
 15. The method of claim 14, wherein the step of removingcomprises passing the human serum albumin composition over anultrafiltration membrane with a molecular weight cut off that retainsthe albumin, and wherein the resulting filtrate comprises DA-DKP. 16.The method of claim 15, wherein the ultrafiltration membrane has amolecular weight cutoff of less than 50 kDa, less than 40 kDa, less than30 kDa, less than 20 kDa, less than 10 kDa, less than 5 kDa or less than3 kDa.
 17. The method of claim 13, wherein the composition furthercomprises NAT, caprylic acid, caprylate or combinations thereof.
 18. Themethod of claim 17, wherein the composition is a composition having aconcentration of NAT, caprylic acid, caprylate or combinations thereoffrom about 4 mM to about 20 mM.
 19. The method of claim 1, wherein thepharmaceutical composition further comprises a second drug selected fromthe group consisting of an analgesic, an anti-inflammatory drug, andcombinations thereof.
 20. The method of claim 1, wherein the number ofdoses in the multi-dose regimen is
 5. 21. The method of claim 1, whereinthe time between doses in the multi-dose regimen is between 2 weeks and12 weeks.
 22. The method of claim 1, wherein the time between doses inthe multi-dose regimen is between 2 weeks and 8 weeks.
 23. The method ofclaim 1, wherein the time between doses in the multi-dose regimen isbetween 2 weeks and 6 weeks.
 24. The method of claim 1, wherein the timebetween doses in the multi-dose regimen is between 2 weeks and 4 weeks.25. The method of claim 1, wherein the time between doses in themulti-dose regimen is between 1 week and 3 weeks.
 26. The method ofclaim 1, wherein the time between doses in the multi-dose regimen aboutis 2 weeks.
 27. The method of claim 1, wherein the amount of each doseis between about 2 mL and about 6 mL.
 28. The method of claim 1, whereinthe amount of each dose is about 4 mL.